Glomerulonephritis biomarkers

ABSTRACT

The present invention relates to methods of diagnosing glomerulonephritis (GN) in a patient, as well as methods of monitoring the progression of GN and/or methods of monitoring a treatment protocol of a therapeutic agent or a therapeutic regimen. The invention also relates to assay methods used in connection with the diagnostic methods described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of U.S. Provisional ApplicationNo. 61/759,434 filed on Feb. 1, 2013, the entire contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

This application relates to assay methods useful in the detection andtreatment of glomerulonephritis.

BACKGROUND OF THE INVENTION

Glomerulonephritis, also known as glomerular nephritis, abbreviated GN,is a renal disease (usually of both kidneys) characterized byinflammation of the glomeruli, or small blood vessels in the kidneys. GNis a type of renal disease in which the part of the kidneys that filterwaste and fluids from the blood is damaged. Damage to the glomerulicauses blood and protein to be lost in the urine. While the exact causeof GN can be difficult to determine, it can be caused by immunologicalproblems. GN can develop quickly, and kidney function can be lost withinweeks or months (called rapidly progressive GN). A quarter of peoplewith chronic GN have no history of kidney disease, but certainconditions can increase an individual's risk of developing GN, includingbut not limited to, blood or lymphatic system disorders, exposure tohydrocarbon solvents, and a history of cancer, infections such as strepinfections, viruses, heart infections, or abscesses. Many conditionscause or increase the risk for GN, including amyloidosis,anti-glomerular basement membrane antibody disease, blood vesseldiseases, such as vasculitis or polyarteritis, focal segmentalglomerulosclerosis, goodpasture syndrome, heavy use of pain relievers,especially NSAIDs, Henoch-Schonlein purpura, IgA nephropathy, Lupusnephritis, and membranoproliferative GN.

SUMMARY OF THE INVENTION

The invention provides a method for evaluating the efficacy of atreatment regimen in a patient diagnosed with glomerulonephritis (GN),said method comprising

(a) obtaining a test sample from a patient undergoing said treatmentregimen for GN;

(b) measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin,Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin, Osteoactin, Albumin,B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin,PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, and combinations thereof;

(c) comparing said level to a normal control level of said biomarker;and

(d) evaluating from said comparing step (c) whether said patient isresponsive to said treatment regimen.

In one embodiment, the invention includes a method for evaluating theefficacy of a treatment regimen in a patient diagnosed withglomerulonephritis (GN), said method comprising

(a) ordering a test comprising a measurement of a level of a biomarkerin a test sample obtained from a patient undergoing said treatmentregimen for GN, wherein said biomarker comprises Clusterin, KIM-1, aGST,IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin,Osteoactin, Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR,LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, andcombinations thereof;

(b) comparing said level to a normal control level of said biomarker;and

(c) evaluating from said comparing step (b) whether said patient isresponsive to said treatment regimen.

Moreover, the invention provides a method of administering a treatmentregimen to a patient in need thereof for treating glomerulonephritis(GN), comprising:

(a) obtaining a test sample from a patient undergoing said treatmentregimen for GN;

(b) measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin,Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin, Osteoactin, Albumin,B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin,PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, and combinations thereof;

(c) comparing said level to a normal control level of said biomarker;

(d) evaluating from said comparing step (c) whether said patient isresponsive to said treatment regimen; and

(e) adjusting said treatment regimen based on said evaluating step (d).

In addition, the invention contemplates a method of administering atreatment regimen to a patient in need thereof for treatingglomerulonephritis (GN), comprising:

(a) obtaining a test sample from a patient prior to the commencement ofsaid treatment regimen for GN;

(b) measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin,Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin, Osteoactin, Albumin,B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin,PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, and combinations thereof;

(c) comparing said level to a normal control level of said biomarker;

(d) evaluating from said comparing step (c) whether said patient will beresponsive to said treatment regimen; and

(e) administering said treatment regimen based on said evaluating step(d).

Still further, the invention includes a method of administering atreatment regimen to a patient in need thereof for treatingglomerulonephritis (GN), comprising:

(a) evaluating a level of a biomarker in a test sample obtained from apatient undergoing said treatment regimen for GN relative to a normalcontrol level of said biomarker, wherein said biomarker comprisesClusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII,UMOD, OPGN, Calbindin, Osteoactin, Albumin, B2M, Cystatin C, NGAL,MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1,VCAM-1, EGF, SERPINB3, and combinations thereof; and

(b) adjusting said treatment regimen based on said evaluating step (a).

Also provided is a method of administering a treatment regimen to apatient in need thereof for treating glomerulonephritis (GN),comprising:

(a) evaluating a level of a biomarker in a test sample obtained from apatient prior to the commencement of said treatment regimen for GNrelative to a normal control level of said biomarker, wherein saidbiomarker comprises Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin,Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin, Osteoactin, Albumin,B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin,PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, and combinations thereof;and

(b) administering said treatment regimen based on said evaluating step(a).

Moreover, the invention provides a multiplexed assay kit used toevaluate the efficacy of a treatment regimen in a patient diagnosed withglomerulonephritis (GN), said kit is configured to measure a level of aplurality of biomarkers in a patient sample, said plurality ofbiomarkers comprises Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin,Timp-1, TNF-RI, TNF-RII, UMOD, and combinations thereof.

In one embodiment, a kit is provided for the analysis of a kidneydisease panel comprising

(a) a multi-well assay plate comprising a plurality of wells, each wellcomprising at least four discrete binding domains to which captureantibodies to the following human analytes are bound: Clusterin, KIM-1,aGST, IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, andcombinations thereof;

(b) in one or more vials, containers, or compartments, a set of labeleddetection antibodies specific for said human analytes; and

(c) in one or more vials, containers, or compartments, a set ofcalibrator proteins.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows elected urine biomarkers with elevated median levels in GNrelative to patients with normal kidney function (N).

FIG. 2 shows biomarkers with lower median levels in GN patients relativeto patients with normal kidney function (N) (biomarkers measured inurine samples are designated by the name of the biomarker followed bythe designation “-u”, e.g., EGF-u, UMOD-u, SerpinB3-u and ANXA1-u andserum UMOD-s).

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The articles“a” and “an” are used herein to refer to one or to more than one (i.e.,to at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element.

As used herein, the term “sample” is intended to mean any biologicalfluid, cell, tissue, organ or combinations or portions thereof, whichincludes or potentially includes a biomarker of a disease of interest.For example, a sample can be a histologic section of a specimen obtainedby biopsy, or cells that are placed in or adapted to tissue culture. Asample further can be a subcellular fraction or extract, or a crude orsubstantially pure nucleic acid molecule or protein preparation. In oneembodiment, the samples that are analyzed in the assays of the presentinvention are blood, peripheral blood mononuclear cells (PBMC), isolatedblood cells, serum and plasma. Other suitable samples include biopsytissue, intestinal mucosa, saliva, cerebral spinal fluid, and urine. Ina preferred embodiment, samples used in the assays of the invention areserum samples.

A “biomarker” is a substance that is associated with a particulardisease. A change in the levels of a biomarker may correlate with therisk or progression of a disease or with the susceptibility of thedisease to a given treatment. A biomarker may be useful in the diagnosisof disease risk or the presence of disease in an individual, or totailor treatments for the disease in an individual (choices of drugtreatment or administration regimes and/or to predict responsiveness ornon-responsiveness to a particular therapeutic regimen). In evaluatingpotential drug therapies, a biomarker may be used as a surrogate for anatural endpoint such as survival or irreversible morbidity. If atreatment alters a biomarker that has a direct connection to improvedhealth, the biomarker serves as a “surrogate endpoint” for evaluatingclinical benefit. A sample that is assayed in the diagnostic methods ofthe present invention may be obtained from any suitable patient,including but not limited to a patient suspected of having GN or apatient having a predisposition to GN. The patient may or may notexhibit symptoms associated with one or more of these conditions.

“Level” refers to the amount, concentration, or activity of a biomarker.The term “level” may also refer to the rate of change of the amount,concentration or activity of a biomarker. A level can be represented,for example, by the amount or synthesis rate of messenger RNA (mRNA)encoded by a gene, the amount or synthesis rate of polypeptidecorresponding to a given amino acid sequence encoded by a gene, or theamount or synthesis rate of a biochemical form of a biomarkeraccumulated in a cell, including, for example, the amount of particularpost-synthetic modifications of a biomarker such as a polypeptide,nucleic acid or small molecule. The term can be used to refer to anabsolute amount of a biomarker in a sample or to a relative amount ofthe biomarker, including amount or concentration determined understeady-state or non-steady-state conditions. Level may also refer to anassay signal that correlates with the amount, concentration, activity orrate of change of a biomarker. The level of a biomarker can bedetermined relative to a control marker or an additional biomarker in asample.

As shown in Table 1, certain biomarkers were found to be altered inserum and urine GN patient samples:

TABLE 1 Sample Change relative to type normal control Biomarker(s) Serumelevated Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin, Timp-1, TNF-RIand TNF-RII. decreased UMOD Urine elevated OPGN, Calbindin, Clusterin,Osteoactin, Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-6, IL-15, CHGA,E-Cadherin, ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1,TNF-RI, TNF-RII and VCAM-1 decreased EGF, UMOD, and SERPINB3

In addition to the serum and urine biomarker levels, fractionalexcretion (FE) was assessed as a measure of the rate of excretion of thebiomarker [FE=U/S[analyte]/U/S[creatinine]×100%]. Using FE, thefollowing biomarkers were identified as having altered excretion ratesin GN patients: OPGN, Calbindin, Clusterin, Osteoactivin, TFF3, B2M,Cystatin C, EGF, NGAL, MCP-1, IL-6, IL-8, IL-15, IL-7, SERPINB3, CHGA,ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, TNF-R1,TNF-RII and VCAM-1.

One or more of the biomarkers listed above can be used for the diagnosisof GN and/or to assess susceptibility of GN in a patient to a treatmentregimen. For example, these biomarkers can be used in a diagnosticmethod, alone or in combination with other biomarkers for GN and/ordiagnostic tests for GN, to diagnose GN in a patient. Alternatively oradditionally, these biomarkers can be used to monitor a therapeuticregimen used for the treatment of GN to assess the efficacy of theregimen for a given patient.

The method of the present invention can include assessing the efficacyof a therapeutic regimen for GN and/or the susceptibility of a patientto a therapeutic regimen. In general, the goal of treatment for GN is toprotect the kidneys from further damage and accordingly, one or more ofthe biomarkers listed above can be analyzed in combination with one ormore biomarkers indicative of renal function, including but not limitedto Lipocalin-2, osteopontin, albumin, TIM-1, NGAL, alpha GST, calbindin,clusterin, KIM-1, osteoactivin, TFF3, VEGF, cystatin C, EGF, OPN, andUMOD. Controlling a patient's blood pressure is critical to protectingthe kidneys and slow the decline of kidney function. Accordingly,diuretics, angiotensin-converting enzyme (ACE) inhibitors, and/orangiotensin II receptor agonists can be used to protect the kidneys fromfurther damage. If an underlying cause of GN has been identified,additional therapeutic agents can be administered, alone or incombination with one or more anti-hypertensive agents, including but notlimited to: antibiotics (if GN resulted from a bacterial infection),corticosteroids and immunosuppressants (if GN resulted from Lupus orvasculitis), fish oil supplements and immunosuppressants (if GN resultedfrom IgA nephropathy), and plasmapheresis (if GN resulted fromGoodpasture's syndrome). The biomarkers identified herein can beassessed before, during and/or after a treatment regimen including oneor more such medications to assess disease progression.

Moreover, a therapeutic regimen may include administration of atherapeutic agent or a combination of therapeutic agents to a patientone or more times over a given time period. This treatment regimen maybe accompanied by the administration of one or more additionaltherapeutic or palliative agents. The level(s) of biomarkers may bemeasured before treatment, one or more times during the administrationperiod, and/or after treatment is suspended. Therefore, the method mayinclude measuring an interim level of a biomarker during the therapeuticregimen and the method includes evaluating biomarker levels by comparingthat level, the interim level and the baseline level. In addition, thelevel of a biomarker may be determined at any time point before and/orafter initiation of treatment. In one embodiment, the biomarker is usedto gauge the efficacy of a therapeutic regimen. Therefore, the method ofthe present invention may include measuring a baseline level(s) of abiomarker before a therapeutic regimen is initiated, and the methodincludes evaluating biomarker levels by comparing the level and thebaseline level.

Still further, the method can include measuring a level(s) of abiomarker before a therapeutic regimen is initiated to predict whetherGN will be responsive or non-responsive to a given therapeutic regimen.The method may further comprise modifying the therapeutic regimen basedon the level(s) of a biomarker observed during this preliminary and/orinterim measuring step, e.g., increasing or decreasing the dosage,frequency, or route of administration of a therapeutic agent, adding anadditional therapeutic agent and/or palliative agent to a treatmentregimen, or if the therapeutic regimen includes the administration oftwo or more therapeutic and/or palliative agents, the treatment regimenmay be modified to eliminate one or more of the therapeutic and/orpalliative agents used in the combination therapy.

Still further, the method can include comparing the level of a biomarkerto a detection cut-off level, wherein a level above the detectioncut-off level is indicative of GN. Alternatively, the evaluating stepcomprises comparing a level of a biomarker to a detection cut-off level,wherein a level below the detection cut-off level is indicative of GN.In one embodiment of the present invention, the level of a biomarker iscompared to a detection cut-off level or range, wherein the biomarkerlevel above or below the detection cut-off level (or within thedetection cut-off range) is indicative of GN. Furthermore, the levels oftwo or more biomarkers may both be used to make a determination. Forexample, i) having a level of at least one of the markers above or belowa detection cut-off level (or within a detection cut-off range) for thatmarker is indicative of GN; ii) having the level of two or more (or all)of the markers above or below a detection cut-off level (or within adetection cut-off range) for each of the markers is indicative of GN; oriii) an algorithm based on the levels of the multiple markers is used todetermine if GN is present.

The methods of the invention can be used alone or in combination withother diagnostic tests or methods to diagnose a patient with GN. Thefollowing tests/criteria are generally used by clinicians to diagnose apatient with GN, and this set of tests can be considered in combinationwith a diagnostic screen for the biomarkers identified herein todiagnose a patient with GN:

-   -   Clinical symptoms: anemia, high blood pressure, reduced kidney        function, signs of chronic kidney disease may be seen,        including: nerve inflammation (polyneuropathy), signs of fluid        overload, including abnormal heart and lung sounds, edema;    -   Imaging tests: abdominal CT scan, kidney ultrasound, chest        x-ray, intravenous pyelogram (IVP)    -   Urinalysis and other urine tests include: creatinine clearance,        urine fortotal protein, uric acid in the urine; urine        concentration test, urine creatinine, urine protein, urine RBC,        urine specific gravity, urine osmolality    -   Blood tests: albumin, anti-glomerular basement membrane antibody        test, anti-neutrophil cytoplasmic antibodies (ANCAs),        anti-nuclear antibodies, BUN and creatinine.

As described herein, the measured levels of one or more biomarkers maybe used to detect or monitor GN and/or to determine the responsivenessof GN to a specific treatment regimen. The specific methods/algorithmsfor using biomarker levels to make these determinations, as describedherein, may optionally be implemented by software running on a computerthat accepts the biomarker levels as input and returns a report with thedeterminations to the user. This software may run on a standalonecomputer or it may be integrated into the software/computing system ofthe analytical device used to measure the biomarker levels or,alternatively, into a laboratory information management system (LIMS)into which crude or processed analytical data is entered. In oneembodiment, biomarkers are measured in a point-of-care clinical devicewhich carries out the appropriate methods/algorithms for detecting,monitoring or determining the responsiveness of a disease and whichreports such determination(s) back to the user.

According to one aspect of the invention, the level(s) of biomarker(s)are measured in samples collected from individuals clinically diagnosedwith, suspected of having or at risk of developing GN. Initial diagnosismay be carried out using conventional methods. The level(s) ofbiomarker(s) are also measured in healthy individuals. Specificbiomarkers valuable in distinguishing between normal and diseasedpatients are identified by visual inspection of the data, for example,by visual classification of data plotted on a one-dimensional ormultidimensional graph, or by using statistical methods such ascharacterizing the statistically weighted difference between controlindividuals and diseased patients and/or by using Receiver OperatingCharacteristic (ROC) curve analysis. A variety of suitable methods foridentifying useful biomarkers and setting detectionthresholds/algorithms are known in the art and will be apparent to theskilled artisan.

For example and without limitation, diagnostically valuable biomarkersmay be first identified using a statistically weighted differencebetween control individuals and diseased patients, calculated as

$\frac{D - N}{\sqrt{\sigma_{D}*\sigma_{N}}}$

wherein D is the median level of a biomarker in patients diagnosed ashaving, for example, GN, N is the median (or average) of the controlindividuals, σ_(D) is the standard deviation of D and σ_(N) is thestandard deviation of N. The larger the magnitude, the greater thestatistical difference between the diseased and normal populations.

According to one embodiment of the invention, biomarkers resulting in astatistically weighted difference between control individuals anddiseased patients of greater than, e.g., 1, 1.5, 2, 2.5 or 3 could beidentified as diagnostically valuable markers.

Another method of statistical analysis for identifying biomarkers is theuse of z-scores, e.g., as described in Skates et al. (2007) CancerEpidemiol. Biomarkers Prey. 16(2):334-341.

Another method of statistical analysis that can be useful in theinventive methods of the invention for determining the efficacy ofparticular candidate analytes, such as particular biomarkers, for actingas diagnostic marker(s) is ROC curve analysis. An ROC curve is agraphical approach to looking at the effect of a cut-off criterion,e.g., a cut-off value for a diagnostic indicator such as an assay signalor the level of an analyte in a sample, on the ability of a diagnosticto correctly identify positive or negative samples or subjects. One axisof the ROC curve is the true positive rate (TPR, i.e., the probabilitythat a true positive sample/subject will be correctly identified aspositive, or alternatively, the false negative rate (FNR=1-TPR, theprobability that a true positive sample/subject will be incorrectlyidentified as a negative). The other axis is the true negative rate,i.e., TNR, the probability that a true negative sample will be correctlyidentified as a negative, or alternatively, the false positive rate(FPR=1-TNR, the probability that a true negative sample will beincorrectly identified as positive). The ROC curve is generated usingassay results for a population of samples/subjects by varying thediagnostic cut-off value used to identify samples/subjects as positiveor negative and plotting calculated values of TPR or FNR and TNR or FPRfor each cut-off value. The area under the ROC curve (referred to hereinas the AUC) is one indication of the ability of the diagnostic toseparate positive and negative samples/subjects. In one embodiment, abiomarker provides an AUC≧0.7. In another embodiment, a biomarkerprovides an AUC≧0.8. In another embodiment, a biomarker provides anAUC≧0.9.

Diagnostic indicators analyzed by ROC curve analysis may be a level ofan analyte, e.g., a biomarker, or an assay signal. Alternatively, thediagnostic indicator may be a function of multiple measured values, forexample, a function of the level/assay signal of a plurality ofanalytes, e.g., a plurality of biomarkers, or a function that combinesthe level or assay signal of one or more analytes with a patient'sscoring value that is determined based on visual, radiological and/orhistological evaluation of a patient. The multi-parameter analysis mayprovide more accurate diagnosis relative to analysis of a single marker.

Candidates for a multi-analyte panel could be selected by using criteriasuch as individual analyte ROC areas, median difference between groupsnormalized by geometric interquartile range (IQR) etc. The objective isto partition the analyte space to improve separation between groups (forexample, normal and disease populations) or to minimize themisclassification rate.

One approach is to define a panel response as a weighted combination ofindividual analytes and then compute an objective function like ROCarea, product of sensitivity and specificity, etc. See e.g., WO2004/058055, as well as US2006/0205012, the disclosures of which areincorporated herein by reference in their entireties.

The assays of the present invention may be conducted by any suitablemethod. In one embodiment, biomarker levels are measured in a singlesample, and those measurement may be conducted in a single assay chamberor assay device, including but not limited to a single well of an assayplate, a single assay cartridge, a single lateral flow device, a singleassay tube, etc. Biomarker levels may be measured using any of a numberof techniques available to the person of ordinary skill in the art,e.g., direct physical measurements (e.g., mass spectrometry) or bindingassays (e.g., immunoassays, agglutination assays andimmunochromatographic assays). The method may also comprise measuring asignal that results from a chemical reactions, e.g., a change in opticalabsorbance, a change in fluorescence, the generation ofchemiluminescence or electrochemiluminescence, a change in reflectivity,refractive index or light scattering, the accumulation or release ofdetectable labels from the surface, the oxidation or reduction or redoxspecies, an electrical current or potential, changes in magnetic fields,etc. Suitable detection techniques may detect binding events bymeasuring the participation of labeled binding reagents through themeasurement of the labels via their photoluminescence (e.g., viameasurement of fluorescence, time-resolved fluorescence, evanescent wavefluorescence, up-converting phosphors, multi-photon fluorescence, etc.),chemiluminescence, electrochemiluminescence, light scattering, opticalabsorbance, radioactivity, magnetic fields, enzymatic activity (e.g., bymeasuring enzyme activity through enzymatic reactions that cause changesin optical absorbance or fluorescence or cause the emission ofchemiluminescence). Alternatively, detection techniques may be used thatdo not require the use of labels, e.g., techniques based on measuringmass (e.g., surface acoustic wave measurements), refractive index (e.g.,surface plasmon resonance measurements), or the inherent luminescence ofan analyte.

Binding assays for measuring biomarker levels may use solid phase orhomogenous formats. Suitable assay methods include sandwich orcompetitive binding assays. Examples of sandwich immunoassays aredescribed in U.S. Pat. No. 4,168,146 and U.S. Pat. No. 4,366,241, bothof which are incorporated herein by reference in their entireties.Examples of competitive immunoassays include those disclosed in U.S.Pat. No. 4,235,601, U.S. Pat. No. 4,442,204 and U.S. Pat. No. 5,208,535,each of which are incorporated herein by reference in their entireties.

Multiple biomarkers may be measured using a multiplexed assay format,e.g., multiplexing through the use of binding reagent arrays,multiplexing using spectral discrimination of labels, multiplexing offlow cytometric analysis of binding assays carried out on particles,e.g., using the Luminex® system. Suitable multiplexing methods includearray based binding assays using patterned arrays of immobilizedantibodies directed against the biomarkers of interest. Variousapproaches for conducting multiplexed assays have been described (Seee.g., US 20040022677; US 20050052646; US 20030207290; US 20030113713; US20050142033; and US 20040189311, each of which is incorporated herein byreference in their entireties. One approach to multiplexing bindingassays involves the use of patterned arrays of binding reagents, e.g.,U.S. Pat. Nos. 5,807,522 and 6,110,426; Delehanty J-B., Printingfunctional protein microarrays using piezoelectric capillaries, MethodsMol. Bio. (2004) 278: 135-44; Lue R Y et al., Site-specificimmobilization of biotinylated proteins for protein microarray analysis,Methods Mol. Biol. (2004) 278: 85-100; Lovett, Toxicogenomics:Toxicologists Brace for Genomics Revolution, Science (2000) 289:536-537; Berns A, Cancer: Gene expression in diagnosis, nature (2000),403, 491-92; Walt, Molecular Biology: Bead-based Fiber-Optic Arrays,Science (2000) 287: 451-52 for more details). Another approach involvesthe use of binding reagents coated on beads that can be individuallyidentified and interrogated. See e.g., WO 9926067, which describes theuse of magnetic particles that vary in size to assay multiple analytes;particles belonging to different distinct size ranges are used to assaydifferent analytes. The particles are designed to be distinguished andindividually interrogated by flow cytometry. Vignali has described amultiplex binding assay in which 64 different bead sets ofmicroparticles are employed, each having a uniform and distinctproportion of two dyes (Vignali, D. A A, “Multiplexed Particle-BasedFlow Cytometric Assays” J. ImmunoL Meth. (2000) 243: 243-55). A similarapproach involving a set of 15 different beads of differing size andfluorescence has been disclosed as useful for simultaneous typing ofmultiple pneumococcal serotypes (Park, M. K et al., “A Latex Bead-BasedFlow Cytometric Immunoassay Capable of Simultaneous Typing of MultiplePneumococcal Serotypes (Multibead Assay)” Clin. Diag. Lab ImmunoL (2000)7: 4869). Bishop, J E et al. have described a multiplex sandwich assayfor simultaneous quantification of six human cytokines (Bishop, LE. etal., “Simultaneous Quantification of Six Human Cytokines in a SingleSample Using Microparticle-based Flow Cytometric Technology,” Clin. Chem(1999) 45:1693-1694).

A diagnostic test may be conducted in a single assay chamber, such as asingle well of an assay plate or an assay chamber that is an assaychamber of a cartridge. The assay modules, e.g., assay plates orcartridges or multi-well assay plates), methods and apparatuses forconducting assay measurements suitable for the present invention aredescribed for example, in US 20040022677; US 20050052646; US20050142033; US 20040189311, each of which is incorporated herein byreference in their entireties. Assay plates and plate readers arecommercially available (MULTI-SPOT® and MULTI-ARRAY® plates and SECTOR®instruments, Meso Scale Discovery, a division of Meso Scale Diagnostics,LLC, Rockville, Md.).

The present invention relates to a kit for the analysis of a panel oftarget analytes. The kit is preferably configured to conduct amultiplexed assay of two or more of the following analytes: Clusterin,KIM-1, aGST, IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD,OPGN, Calbindin, Osteoactin, Albumin, B2M, Cystatin C, NGAL, MCP-1,IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1,VCAM-1, EGF, and SERPINB3, and combinations thereof. The kit can include(a) a single panel arrayed on a multi-well plate which is configured tobe used in an electrochemiluminescence assay, as well as (b) associatedconsumables, e.g., detection antibodies, calibrators, and optionaldiluents and/or buffers. Alternatively, the multi-well plates andassociated consumables can be provided separately.

The panel is preferably configured in a multi-well assay plate includinga plurality of wells, each well having an array with “spots” or discretebinding domains. Preferably, the array includes one, four, seven, ten,sixteen, or twenty-five binding domains, and most preferably, the arrayincludes one, four, seven, or ten binding domains. A capture antibody toeach analyte is immobilized on a binding domain in the well and thatcapture antibody is used to detect the presence of the target analyte inan immunoassay. Briefly, a sample suspected of containing that analyteis added to the well and if present, the analyte binds to the captureantibody at the designated binding domain. The presence of bound analyteon the binding domain is detected by adding labeled detection antibody.The detection antibody also binds to the analyte forming a “sandwich”complex (capture antibody—analyte—detection antibody) on the bindingdomain.

The multiplexed immunoassay kits described herein allow a user tosimultaneously quantify multiple biomarkers. The panels are selected andoptimized such that the individual assays function well together. Thesample may require dilution prior to being assayed. Sample dilutions forspecific sample matrices of interest are optimized for a given panel tominimize sample matrix effects and to maximize the likelihood that allthe analytes in the panel will be within the dynamic range of the assay.In a preferred embodiment, all of the analytes in the panel are analyzedwith the same sample dilution in at least one sample type. In anotherpreferred embodiment, all of the analytes in a panel are measured usingthe same dilution for most sample types.

For a given panel, the detection antibody concentration and the numberof labels per protein (L/P ratio) for the detection antibody areadjusted to bring the expected levels of all analytes into aquantifiable range at the same sample dilution. If one wants to increasethe high end of the quantifiable range for a given analyte, then the L/Pcan be decreased and/or the detection antibody concentration isdecreased. On the other hand, if one wants to increase the lower end ofthe quantifiable range, the L/P can be increased, the detection antibodyconcentration can be increased if it is not at the saturation level,and/or the background signal can be lowered.

Calibration standards for use with the assay panels are selected toprovide the appropriate quantifiable range with the recommended sampledilution for the panel. The calibration standards have knownconcentrations of one of more of the analytes in the panel.Concentrations of the analytes in unknown samples are determined bycomparison to these standards. In one embodiment, calibration standardscomprise mixtures of the different analytes measured by an assay panel.Preferably, the analyte levels in a combined calibrator are selectedsuch that the assay signals for each analyte are comparable, e.g.,within a factor of two, a factor of five or a factor of 10. In anotherembodiment, calibration standards include mixtures of analytes frommultiple different assay panels.

A calibration curve may be fit to the assay signals measured withcalibration standards using, e.g., curve fits known in the art such aslinear fits, 4-parameter logistic (4-PL) and 5-parameter (5-PL) fits.Using such fits, the concentration of analytes in an unknown sample maybe determined by backfitting the measured assay signals to thecalculated fits. Measurements with calibration standards may also beused to determine assay characteristics such as the limit of detection(LOD), limit of quantification (LOQ), dynamic range, and limit oflinearity (LOL).

A kit can include the following assay components: a multi-well assayplate configured to conduct an immunoassay for one of the panelsdescribed herein, a set of detection antibodies for the analytes in thepanel (wherein the set comprises individual detection antibodies and/ora composition comprising a blend of one or more individual detectionantibodies), and a set of calibrators for the analytes in the panel(wherein the set comprises individual calibrator protein compositionsand/or a composition comprising a blend of one or more individualcalibrator proteins). The kit can also include one of more of thefollowing additional components: a blocking buffer (used to block assayplates prior to addition of sample), an antibody diluent (used to dilutestock detection antibody concentrations to the working concentration),an assay diluent (used to dilute samples), a calibrator diluent (used todilute or reconstitute calibration standards) and a read buffer (used toprovide the appropriate environment for detection of assay labels, e.g.,by an ECL measurement). The antibody and assay diluents are selected toreduce background, optimize specific signal, and reduce assayinterference and matrix effect. The calibrator diluent is optimized toyield the longest shelf life and retention of calibrator activity. Theblocking buffer should be optimized to reduce background. The readbuffer is selected to yield the appropriate sensitivity, quantifiablerange, and slowest off-rate.

The reagent components of the kit can be provided as liquid reagents,lyophilized, or combinations thereof, diluted or undiluted, and the kitincludes instructions for appropriate preparation of reagents prior touse. In a preferred embodiment, a set of detection antibodies areincluded in the kit comprising a plurality of individual detectionantibody compositions in liquid form. Moreover, the set of calibratorsprovided in the kit preferably comprise a lyophilized blend ofcalibrator proteins. Still further, the kit includes a multi-well assayplate that has been pre-coated with capture antibodies and exposed to astabilizing treatment to ensure the integrity and stability of theimmobilized antibodies.

As part of a multiplexed panel development, assays are optimized toreduce calibrator and detection antibody non-specific binding. Insandwich immunoassays, specificity mainly comes from capture antibodybinding. Some considerations for evaluating multiplexed panels include:(a) detection antibody non-specific binding to capture antibodies isreduced to lower background of assays in the panel, and this can beachieved by adjusting the concentrations and L/P of the detectionantibodies; (b) non-specific binding of detection antibodies to othercalibrators in the panel is also undesirable and should be minimized;(c) non-specific binding of other calibrators in the panel and otherrelated analytes should be minimized; if there is calibratornon-specific binding, it can reduce the overall specificity of theassays in the panel and it can also yield unreliable results as therewill be calibrator competition to bind the capture antibody.

Different assays in the panel may require different incubation times andsample handling requirements for optimal performance. Therefore, thegoal is to select a protocol that's optimized for most assays in thepanel. Optimization of the assay protocol includes, but is not limitedto, adjusting one or more of the following protocol parameters: timing(incubation time of each step), preparation procedure (calibrators,samples, controls, etc.), and number of wash steps.

The reagents used in the kits, e.g., the detection and captureantibodies and calibrator proteins, are preferably subjected toanalytical testing and meet or exceed the specifications for thosetests. The analytical tests that can be used to characterize kitmaterials include but are not limited to, CIEF, DLS, reducing and/ornon-reducing EXPERION, denaturing SDS-PAGE, non-denaturing SDS-PAGE,SEC-MALS, and combinations thereof. In a preferred embodiment, thematerials are characterized by CIEF, DLS, and reducing and non-reducingEXPERION. One or more additional tests, including but not limited todenaturing SDS-PAGE, non-denaturing SDS-PAGE, SEC-MALS, and combinationsthereof, can also be used to characterize the materials. In a preferredembodiment, the materials are also subjected to functional testing,i.e., a binding assay for the target analyte, as well as one or morecharacterization tests, such as those listed above. If the materials donot meet or exceed the specifications for the functional and/orcharacterization tests, they can be subjected to additional purificationsteps and re-tested. Each of these tests and the metrics applied to theanalysis of raw materials subjected to these tests are described below:

Capillary Isoelectric Focusing (CIEF) is a technique commonly used toseparate peptides and proteins, and it is useful in the detection ofaggregates. During a CIEF separation, a capillary is filled with thesample in solution and when voltage is applied, the ions migrate to aregion where they become neutral (pH=pI). The anodic end of thecapillary sits in acidic solution (low pH), while the cathodic end sitsin basic solution (high pH). Compounds of equal isoelectric points (pI)are “focused” into sharp segments and remain in their specific zone,which allows for their distinct detection based on molecular charge andisoelectric point. Each specific antibody solution will have afingerprint CIEF that can change over time. When a protein solutiondeteriorates, the nature of the protein and the charge distribution canchange. Therefore, CIEF is a particularly useful tool to assess therelative purity of a protein solution and it is a preferred method ofcharacterizing the antibodies and calibrators in the plates and kitsdescribed herein. The metrics used in CIEF include pI of the main peak,the pI range of the solution, and the profile shape, and each of thesemeasurements are compared to that of a reference standard.

Dynamic Light Scattering (DLS) is used to probe the diffusion ofparticulate materials either in solution or in suspension. Bydetermining the rate of diffusion (the diffusion coefficient),information regarding the size of particles, the conformation ofmacromolecular chains, various interactions among the constituents inthe solution or suspension, and even the kinetics of the scatterers canbe obtained without the need for calibration. In a DLS experiment, thefluctuations (temporal variation, typically in a μs to ms time scale) ofthe scattered light from scatterers in a medium are recorded andanalyzed in correlation delay time domain. Like CIEF, each proteinsolution will generate a fingerprint DLS for the particle size and it'sideally suited to detect aggregation. All IgGs, regardless of bindingspecificity, will exhibit the same DLS particle size. The metrics usedto analyze a protein solution using DLS include percentagepolydispersity, percentage intensity, percentage mass, and the radius ofthe protein peak. In a preferred embodiment, an antibody solution meetsor exceeds one or more of the following DLS specifications: (a) radiusof the antibody peak: 4-8 nm (antibody molecule size); (b)polydispersity of the antibody peak: <40% (measure of size heterogeneityof antibody molecules); (c) intensity of the antibody peak: >50% (ifother peaks are present, then the antibody peak is the predominantpeak); and (d) mass in the antibody peak: >50%.

Reducing and non-reducing gel electrophoresis are techniques well knownin the art. The EXPERION™ (Bio-Rad Laboratories, Inc., www.bio-rad.com)automated electrophoresis station performs all of the steps of gel-basedelectrophoresis in one unit by automating and combining electrophoresis,staining, destaining, band detection, and imaging into a single step. Itcan be used to measure purity. Preferably, an antibody preparation isgreater 50% pure by Experion, more preferably, greater than 75% pure,and most preferably greater than 80% pure. Metrics that are applied toprotein analysis using non-reducing Experion include percentage totalmass of protein, and for reducing Experion they include percentage totalmass of the heavy and light chains in an antibody solution, and theheavy to light chain ratio.

Multi-Angle Light Scattering (MALS) detection can be used in thestand-alone (batch) mode to measure specific or non-specific proteininteractions, as well as in conjunction with a separation system such asflow field flow fractionation (FFF) or size exclusion chromatography(SEC). The combined SEC-MALS method has many applications, such as theconfirmation of the oligomeric state of a protein, quantification ofprotein aggregation, and determination of protein conjugatestoichiometry. Preferably, this method is used to detect molecularweight of the components of a sample.

As used herein, a lot of kits comprise a group of kits comprising kitcomponents that meet a set of kit release specifications. A lot caninclude at least 10, at least 100, at least 500, at least 1,000, atleast 5,000, or at least 10,000 kits and a subset of kits from that lotare subjected to analytical testing to ensure that the lot meets orexceeds the release specifications. In one embodiment, the releasespecifications include but are not limited to kit processing, reagentstability, and kit component storage condition specifications. Kitprocessing specifications include the maximum total sample incubationtime and the maximum total time to complete an assay using the kit.Reagent stability specifications include the minimum stability of eachreagent component of the kit at a specified storage temperature. Kitstorage condition specifications include the range of storagetemperatures for all components of the kit, the maximum storagetemperature for frozen components of the kit, and the maximum storagetemperature for non-frozen components of the kit. A subset of kits in alot is reviewed in relation to these specifications and the size of thesubset depends on the lot size. In a preferred embodiment, for a lot ofup to 300 kits, a sampling of 4-7 kits are tested; for a lot of 300-950kits, a sampling of 8-10 kits are tested; and for a lot of greater than950 kits, a sampling of 10-12 kits are tested. Alternatively oradditionally, a sampling of up to 1-5% preferably up to 1-3%, and mostpreferably up to 2% is tested.

In addition, each lot of multi-well assay plates is preferably subjectedto uniformity and functional testing. A subset of plates in a lot issubjected to these testing methods and the size of the subset depends onthe lot size. In a preferred embodiment, for a lot of up to 300 plates,a sampling of 4-7 plates are tested; for a lot of 300-950 plates, asampling of 8-10 plates are tested; and for a lot of greater than 950plates, a sampling of 10-12 plates are tested. Alternatively oradditionally, a sampling of up to 1-5% preferably up to 1-3%, and mostpreferably up to 2% is tested. The uniformity and functional testingspecifications are expressed in terms of % CV, Coefficient ofVariability, which is a dimensionless number defined as the standarddeviation of a set of measurements, in this case, the relative signaldetected from binding domains across a plate, divided by the mean of theset.

One type of uniformity testing is protein A/G testing. Protein A/Gbinding is used to confirm that all binding domains within a plate arecoupled to capture antibody. Protein A/G is a recombinant fusion proteinthat combines IgG binding domains of Protein A and protein G and itbinds to all subclasses of human IgG, as well as IgA, IgE, IgM and, to alesser extent, IgD. Protein A/G also binds to all subclasses of mouseIgG but not mouse IgA, IgM, or serum albumin, making it particularlywell suited to detect mouse monoclonal IgG antibodies withoutinterference from IgA, IgM, and serum albumin that might be present inthe sample matrix. Protein A/G can be labeled with a detectable moiety,e.g., a fluorescent, chemiluminescent, or electrochemiluminescent label,preferably an ECL label, to facilitate detection. Therefore, if captureantibody is adhered to a binding domain of a well, it will bind tolabeled protein A/G, and the relative amount of capture antibody boundto the surface across a plate can be measured.

In addition to the uniformity testing described above, a uniformitymetric for a subset of plates within a lot can be calculated to assesswithin-plate trending. A uniformity metric is calculated using a matrixof normalized signals from protein A/G and/or other uniformity orfunctional tests. The raw signal data is smoothed by techniques known inthe art, thereby subtracting noise from the raw data, and the uniformitymetric is calculated by subtracting the minimum signal in the adjusteddata set from the maximum signal.

In a preferred embodiment, a subset of plates in a lot is subjected toprotein A/G and functional testing and that subset meet or exceed thefollowing specifications:

TABLE 2 Plate Metrics Preferred Specification for a subset Metric of 96well multi-well plates Average intraplate CV ≦10% Maximum intraplate CV≦13% Average Uniformity ≦25% Maximum Uniformity ≦37% CV of intraplateaverages ≦18% Signal, lower boundary >1500 Signal, upper boundary  <10⁽⁶⁾

As disclosed in U.S. Pat. No. 7,842,246 to Wohlstadter et al., thedisclosure of which is incorporated herein by reference in its entirety,each plate consists of several elements, e.g., a plate top, a platebottom, wells, working electrodes, counter electrodes, referenceelectrodes, dielectric materials, electrical connects, and assayreagents. The wells of the plate are defined by holes/openings in theplate top. The plate bottom can be affixed, manually or by automatedmeans, to the plate top, and the plate bottom can serve as the bottom ofthe well. Plates may have any number of wells of any size or shape,arranged in any pattern or configuration, and they can be composed of avariety of different materials. Preferred embodiments of the inventionuse industry standard formats for the number, size, shape, andconfiguration of the plate and wells. Examples of standard formatsinclude 96, 384, 1536, and 9600 well plates, with the wells configuredin two-dimensional arrays. Other formats may include single well plates(preferably having a plurality of assay domains that form spot patternswithin each well), 2 well plates, 6 well plates, 24 well plates, and6144 well plates. Each well of the plate includes a spot pattern ofvarying density, ranging from one spot within a well to 2, 4, 7, 9, 10,16, 25, etc., as described hereinabove.

Each plate is assembled according to a set of preferred specifications.In a preferred embodiment, a plate bottom meets or exceeds the followingspecifications:

TABLE 3 Plate bottom specifications 96-well (round well) Parameterspecifications in inches Length range (C to C)* 3.8904-3.9004 (A1-A12and H1-H12) Width range (C to C) 2.4736-2.4836 (A1-A12 and H1-H12) Wellto well spacing 0.3513-0.3573 *C to C well distance is the center ofspot to center of spot distance between the outermost wells of a plate.

In a further preferred embodiment, the plate also meets or exceedsdefined specifications for alignment of a spot pattern within a well ofthe plate. These specifications include three parameters: (a) Δx, thedifference between the center of the spot pattern and the center of thewell along the x axis of the plate (column-wise, long axis); (b) Δy, thedifference between the center of the spot pattern and the center of thewell along the y axis of the plate (row-wise, short axis); and (c) α,the counter-clockwise angle between the long axis of the plate bottomand the long axis of the plate top of a 96-well plate. In a preferredembodiment, the plate meets or exceeds the following specifications:Δx≦0.2 mm, Δy≦0.2 mm, and α≦0.1°.

The following non-limiting examples serve to illustrate rather thanlimit the present invention.

EXAMPLES Measurement of biomarkers indicative of Glomerulonephritis

Urine and serum samples were collected from 23 patients previouslydiagnosed with GN and 6 normal control subjects (patients having normalkidney function). Samples were analyzed in an immunoassay format and thesamples were screened for the presence/absence of the following set ofbiomarkers: AKR1B1, AKR1C2, Albumin, ALP, ANXA1, B2M, BCL2L2, Calbindin,CHGA, Clusterin, CRYAB, Cystatin C, E-Cadherin, EGF, EOTAXIN, EOTAXIN-3,FABP5, GCLM, GLO1, GM-CSF, GPI, GSTM1, GSTM2, ICAM-1, IFN-gamma,IFN-gamma, IL-10, IL-12P70, IL-13, IL-15, IL-16, IL18, IL-1 alpha, IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, KDR, KIM-1, LBP,MAGEA4, MAPK14, MBD1, MCP-1, MCP-4, MDC, MIP-1B, NGAL, NME2, OCL, ODC1,ONN, OPGN, OPN, osteoactivin, Osteopontin, P-Cadherin, PPP2R4, PRDX4,Prolactin, PSAT1, RAC1, RANTES, S100A4, S100A6, SAT1, SERPINB3, SFN,SOST, TARC, TFF3, TIMP-1, TNF RI, TNF RII, TNF-alpha, UMOD, VCAM-1, VEGFand aGST.

In general, the assay format was as follows, (all consumables, reagents,and instruments referenced below were supplied by Meso Scale Discovery,Rockville, Md.): (1) block MSD MULTI-SPOT® plate for 1 hour withappropriate MSD® blocking solution and wash; (2) add 25 μl assay diluentto each well, if specified; (3) add 25 μl calibrator, or sample (dilutedas appropriate) to each well; (4) incubate with shaking for 1-3 hours(time as specified) and wash the well; (5) add 25 μl labeled detectionantibody solution to each well; (6) incubate with shaking for 1-2 hours(time as specified) and wash the well; (7) add 150 μl MSD read buffer toeach well; (8) read plate immediately on MSD SECTOR® 6000 Reader.

The following biomarkers were elevated in serum samples of GN patientsrelative to normal patients, Clusterin, KIM-1, aGST, IL-6, CHGA,E-Cadherin, Timp-1, TNF-RI and TNF-RII (Table 4). Lower serum levels ofUMOD were also observed, a protein produced in the kidney (Henle's loop)and excreted in the urine, indicating kidney damage (Table 4, FIG. 1).

TABLE 4 Serum biomarkers with altered levels in GN relative to patientswith normal kidney function (N). Median values are in pg/ml in serum.Biomarker Clusterin KIM-1 aGST UMOD IL-6 CHGA E-Cadherin Timp-l TNF-RITNF-RII median GN 57,324,966 587 3,369 49,081 1.62 24,972 68,119 816,3914,740 7,901 median N 25,705,457 211 1,640 122,308 0.41 7,306 26,247258,562 2,004 2,966 medGN/medN 2.23 2.78 2.05 0.40 3.99 3.42 2.60 3.162.37 2.66

In urine samples, a larger number of proteins with altered levels wereidentified, following normalization of these values to urine creatinineto correct for urine volume. The following biomarkers had elevatedmedian values in GN patients: OPGN, Calbindin, Clusterin, Osteoactin,Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-6, IL-15, CHGA, E-Cadherin,ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, TNF-RI,TNF-RII and VCAM-1 (Table 5, FIG. 1). The following biomarkers had lowerlevels, EGF, UMOD, SERPINB3 and ANXA1 (Table 5, FIG. 2).

TABLE 5 Urine biomarkers with altered levels in GN relative to patientswith normal kidney function (N). Median normalized values weredetermined using the urine biomarker levels normalized to urinecreatinine (normalized biomarker value = [pg/ml of biomarker]/[urinecreatinine, mg/dl]. Biomarker OPGN Calbindin Clusterin OsteoactinAlbumin B2M Cystatin C EGF NGAL S100A4 median GN 0.86 87 996 18.573,758,615 14,241 917 50 849 8.25 median N 0.23 31 158 6.74 35,912 697296 102 312 1.85 medGN/medN 3.67 2.78 6.29 2.76 105 20.42 3.10 0.49 2.724.46 Biomarker ANXA1 CHGA E-Cadherin ICAM-1 KDR LBP PRDX4 ProlactinPSAT1 median GN 1.59 0.90 119.24 48.88 1.98 10.02 23.16 1.08 0.35 medianN 4.08 0.14 31.04 20.21 0.15 1.24 8.38 0.05 0.05 medGN/medN 0.39 6.533.84 2.42 13.51 8.06 2.76 23.48 7.20 Biomarker UMOD MCP-1 IL-6 IL-15SERPINB3 TIMP-1 TNF-RI TNF-RII VCAM-1 median GN 70,843 3.03 0.022 0.0172.69 13.88 46.64 91.68 26.70 median N 190,608 0.76 0.005 0.004 7.22 1.7814.01 22.77 7.41 medGN/medN 0.37 3.98 4.56 4.42 0.37 7.79 3.33 4.03 3.61

As expected, the levels of albumin and other proteins were significantlyelevated in urine, indicating damage to the kidney, allowing theseproteins appear at elevated levels in the urine of GN patients. Althougha general elevation of protein levels might have been expected acrossthe biomarkers tested this did not prove to be the case, indicating thatadditional disease-specific diagnostic value was associated with theseelevated biomarkers. In the case of the biomarkers with lower levels inthe GN patients, these are of special interest since they contrast tothe elevated protein levels anticipated in patients with damagedkidneys, and as such are especially valuable biomarkers for evaluatingGN disease.

In addition to the serum and urine biomarker levels, the fractionalexcretion (FE) was calculated as a measure of the rate of excretion of abiomarker [FE=U/S[analyte]/U/S[creatinine]×100%]. Using FE, thefollowing biomarkers were identified as having altered excretion ratesin GN patients, OPGN, Calbindin, Clusterin, Osteoactivin, TFF3, B2M,Cystatin C, EGF, NGAL, MCP-1, IL-6, IL-8, IL-15, IL-7, SERPINB3, CHGA,ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, TNF-R1,TNF-RII and VCAM-1 (Table 6).

TABLE 6 Biomarkers with altered fractional excretion (FE) rates, in GNrelative to patients with normal kidney function (N). Median normalizedvalues were determined using the FE [FE = U/S[analyte]/U/S[creatinine] ×100%]. Biomarker OPGN Calbindin Clusterin Osteoactivin TFF3 B2M CystatinC EGF NGAL median GN 87% 124% 0.42% 10.90% 25.77% 42.53% 40.16%  2892%38% median N 12%  42% 0.05%  1.90%  9.53%  2.87%  2.34% 11812% 12%medGN/medN 7.26 2.94 9.07 5.73 2.70 14.83 17.16 0.24 3.06 BiomarkerMCP-1 IL-6 IL-8 IL-15 IL-7 SERPINB3 CHGA ICAM-1 KDR median GN 102% 372%174% 199% 32%  55% 0.83% 4.58% 1.32% median N  23%  68%  69%  23% 14%223% 0.19% 1.15% 0.07% medGN/medN 4.43 5.50 2.53 8.71 2.21 0.25 4.284.00 17.82 Biomarker LBP PRDX4 Prolactin PSAT1 S100A4 TIMP-1 TNF-R1TNF-RII VCAM-1 median GN 0.36% 12.75% 7.35% 1.31% 4.60% 0.47% 323% 232%1.00% median N 0.03%  4.37% 0.05% 0.38% 0.91% 0.06%  56%  70% 0.20%medGN/medN 13.4 2.9 143.6 3.41 5.06 7.38 5.77 3.32 5.00

Various publications and test methods are cited herein, the disclosuresof which are incorporated herein by reference in their entireties, Incases where the present specification and a document incorporated byreference and/or referred to herein include conflicting disclosure,and/or inconsistent use of terminology, and/or theincorporated/referenced documents use or define terms differently thanthey are used or defined in the present specification, the presentspecification shall control.

REFERENCES

-   1. Appel G B. Glomerular disorders and nephrotic syndromes. In:    Goldman L, Ausiello D, eds. Cecil Medicine. 23rd ed. Philadelphia,    Pa.: Saunders Elsevier; 2007: chap 122.-   2. Nachman P H, Jennette J C, Falk R J. Primary glomerular disease.    In: Brenner B M, ed. Brenner and Rector's The Kidney. 8th ed.    Philadelphia, Pa.: Saunders Elsevier; 2007: chap 30.

1. A method for evaluating the efficacy of a treatment regimen in apatient diagnosed with glomerulonephritis (GN), said method comprising(a) obtaining a test sample from a patient undergoing said treatmentregimen for GN; (b) measuring a level of a biomarker in said testsample, wherein said biomarker comprises Clusterin, KIM-1, aGST, IL-6,CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin,Osteoactin, Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR,LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, andcombinations thereof; (c) comparing said level to a normal control levelof said biomarker; and (d) evaluating from said comparing step (c)whether said patient is responsive to said treatment regimen.
 2. Amethod for evaluating the efficacy of a treatment regimen in a patientdiagnosed with glomerulonephritis (GN), said method comprising (a)ordering a test comprising a measurement of a level of a biomarker in atest sample obtained from a patient undergoing said treatment regimenfor GN, wherein said biomarker comprises Clusterin, KIM-1, aGST, IL-6,CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin,Osteoactin, Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR,LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, andcombinations thereof; (b) comparing said level to a normal control levelof said biomarker; and (c) evaluating from said comparing step (b)whether said patient is responsive to said treatment regimen.
 3. Amethod of administering a treatment regimen to a patient in need thereoffor treating glomerulonephritis (GN), comprising: (a) obtaining a testsample from a patient undergoing said treatment regimen for GN; (b)measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin,Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin, Osteoactin, Albumin,B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin,PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, and combinations thereof;(c) comparing said level to a normal control level of said biomarker;(d) evaluating from said comparing step (c) whether said patient isresponsive to said treatment regimen; and (e) adjusting said treatmentregimen based on said evaluating step (d).
 4. A method of administeringa treatment regimen to a patient in need thereof for treatingglomerulonephritis (GN), comprising: (a) obtaining a test sample from apatient prior to the commencement of said treatment regimen for GN; (b)measuring a level of a biomarker in said test sample, wherein saidbiomarker comprises Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin,Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin, Osteoactin, Albumin,B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin,PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, and combinations thereof;(c) comparing said level to a normal control level of said biomarker;(d) evaluating from said comparing step (c) whether said patient will beresponsive to said treatment regimen; and (e) administering saidtreatment regimen based on said evaluating step (d).
 5. A method ofadministering a treatment regimen to a patient in need thereof fortreating glomerulonephritis (GN), comprising: (a) evaluating a level ofa biomarker in a test sample obtained from a patient undergoing saidtreatment regimen for GN relative to a normal control level of saidbiomarker, wherein said biomarker comprises Clusterin, KIM-1, aGST,IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin,Osteoactin, Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR,LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, andcombinations thereof; and (b) adjusting said treatment regimen based onsaid evaluating step (a).
 6. A method of administering a treatmentregimen to a patient in need thereof for treating glomerulonephritis(GN), comprising: (a) evaluating a level of a biomarker in a test sampleobtained from a patient prior to the commencement of said treatmentregimen for GN relative to a normal control level of said biomarker,wherein said biomarker comprises Clusterin, KIM-1, aGST, IL-6, CHGA,E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, OPGN, Calbindin, Osteoactin,Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-15, ICAM-1, KDR, LBP, PRDX4,Prolactin, PSAT1, S100A4, TIMP-1, VCAM-1, EGF, SERPINB3, andcombinations thereof; and (b) administering said treatment regimen basedon said evaluating step (a).
 7. A method according to claim 1 whereinsaid measuring step comprises conducting a multiplexed assay measurementof a plurality of said biomarkers in said test sample, wherein saidmultiplexed assay measurement is conducted using one reaction volumecomprising said test sample.
 8. The method of claim 1 wherein saidmethod comprises measuring levels of two or more biomarkers.
 9. Themethod of claim 8 wherein said measuring step comprises measuring levelsof a first biomarker and an additional biomarker comprising Clusterin,KIM-1, aGST, IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD,OPGN, Calbindin, Osteoactin, Albumin, B2M, Cystatin C, NGAL, MCP-1,IL-15, ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1,VCAM-1, EGF, SERPINB3, and combinations thereof.
 10. A method of claim 8wherein said first biomarker and said additional biomarker are selectedfrom: Clusterin, KIM-1, aGST, IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI,TNF-RII, and UMOD.
 11. A method of claim 1 further comprising one ormore additional measuring steps including: (x) measuring a baselinelevel(s) of said biomarker before said treatment regimen is initiated,and said evaluating step further comprises comparing said level and saidbaseline level; and (y) measuring an interim level of said biomarkerduring said treatment regimen and said evaluating step further comprisescomparing said level, said interim level and said baseline level. 12.The method of claim 1, wherein said evaluating step comprises comparingsaid level of said biomarker to a detection cut-off level, wherein saidlevel above said detection cut-off level is indicative of GN.
 13. Themethod of claim 1, wherein said evaluating step comprises comparing saidlevel of said biomarker to a detection cut-off level, wherein said levelbelow said detection cut-off level is indicative of GN.
 14. The methodof claim 1 further comprising determining from said level of saidbiomarker the disease progression of GN.
 15. A multiplexed assay kitused to evaluate the efficacy of a treatment regimen in a patientdiagnosed with glomerulonephritis (GN), said kit is configured tomeasure a level of a plurality of biomarkers in a patient sample, saidplurality of biomarkers comprises Clusterin, KIM-1, aGST, IL-6, CHGA,E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, and combinations thereof. 16.The kit of claim 15 wherein said kit is further configured to comparesaid level to a level of a normal control.
 17. The kit of claim 15wherein said kit comprises a multi-well assay plate and wherein a wellof said assay plate comprises a plurality of assay domains, at least twoof said assay domains comprising reagents for measuring differentbiomarkers.
 18. The kit of claim 15 wherein said kit comprises an assaycartridge for conducting a plurality of assays, said cartridgecomprising a flow cell having an inlet, an outlet or a detectionchamber, said inlet, detecting chamber, or outlet defining a flow paththrough said flow cell, said detection chamber configured to measuresaid level of said plurality of biomarkers in said sample.
 19. The kitof claim 15 wherein said kit further comprises one or more additionalassay reagents used in said assay, said one or more additional assayreagents provided in one or more vials, containers, or compartments ofsaid kit.
 20. A kit for the analysis of a kidney disease panelcomprising (a) a multi-well assay plate comprising a plurality of wells,each well comprising at least four discrete binding domains to whichcapture antibodies to the following human analytes are bound: Clusterin,KIM-1, aGST, IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, andcombinations thereof; (b) in one or more vials, containers, orcompartments, a set of labeled detection antibodies specific for saidhuman analytes; and (c) in one or more vials, containers, orcompartments, a set of calibrator proteins.
 21. The kit of claim 20wherein said kit further comprises one or more diluents.
 22. The kit ofclaim 20 wherein said detection antibodies are labeled with anelectrochemiluminescent (ECL) label.
 23. The kit of claim 22 whereinsaid kit further comprises an ECL read buffer.
 24. The kit of claim 20wherein said discrete binding domains are positioned on an electrodewithin said well.
 25. The kit of claim 20 wherein said set of calibratorproteins comprise a lyophilized blend of proteins.
 26. The kit of claim20 wherein said set of calibrator proteins comprise a liquid formulationof calibrator proteins.
 27. The method of claim 1, wherein said samplecomprises serum and said biomarker comprises Clusterin, KIM-1, aGST,IL-6, CHGA, E-Cadherin, Timp-1, TNF-RI, TNF-RII, UMOD, and combinationsthereof.
 28. The method of claim 1, wherein said sample comprises urineand said biomarker comprises OPGN, Calbindin, Clusterin, Osteoactin,Albumin, B2M, Cystatin C, NGAL, MCP-1, IL-6, IL-15, CHGA, E-Cadherin,ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, TNF-RI,TNF-RII, VCAM-1, EGF, UMOD, SERPINB3, and combinations thereof.
 29. Themethod of claim 1 wherein said sample comprises serum and/or urine andsaid biomarker comprises OPGN, Calbindin, Clusterin, Osteoactivin, TFF3,B2M, Cystatin C, EGF, NGAL, MCP-1, IL-6, IL-8, IL-15, IL-7, SERPINB3,CHGA, ICAM-1, KDR, LBP, PRDX4, Prolactin, PSAT1, S100A4, TIMP-1, TNF-R1,TNF-RII, VCAM-1 and combinations thereof.
 30. The method of claim 29wherein said method further comprises determining the fractionalexcretion of said biomarker.